Hepatitis C virus (HCV) remains a major threat to public health despite the advent of direct acting antivirals (DAAs) targeting viral protease and RNA polymerase. Resistance development, as a natural outcome from attack by therapeutics, and the prohibitive cost of DAAs demand alternative and more powerful therapeutics. Our laboratory has been studying the mechanisms of HCV entry in the hopes of identifying novel cellular targets for antiviral development. Entry inhibitors targeting host functions will be particularly valuable in treating post-transplant hepatitis C, where one of the major unmet needs is the development of strategies to prevent re- infection of the liver graft after transplantation. During the previous funding period, we found that cell signaling proteins prohibitins 1 and 2 (PHB1/2) are essential pan-genomic HCV entry factors that function at a post- binding step. Although commonly known as mitochondrial inner membrane proteins, a fraction of endogenous PHB1 and 2 localize to the cell surface. Interestingly, plasma-membrane-associated PHBs are known for membrane targeting and activation of CRaf by Ras upon epidermal growth factor receptor (EGFR) activation. Targeting PHBs by rocaglamide (Roc-A), a natural compound that binds both PHB1/2, reduced cell surface PHB1/2, disrupted PHB-CRaf interaction, and inhibited HCV entry at low nanomolar concentrations. A subsequent screen of a collection of synthetic Roc-A derivatives indicated that the (-) Roc-A enantiomer is a more potent entry inhibitor than the (+) Roc-A enantiomer. Strikingly, the chiral, racemic version of a Roc-A derivative displayed improved potency and therapeutic index against HCV infection in cell culture. These findings open an unprecedented opportunity to dissect the HCV entry process and to translate basic research into antiviral development. In this competing renewal, we propose to dissect the master signaling pathway that coordinates HCV entry and optimize our lead compound for treating HCV infection. Accomplishing the project will significantly advance our understanding of HCV entry, and lead to a new class of entry inhibitors.